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In JoVE (1)
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Articles by John H. Horne in JoVE
JoVE Neuroscience
מיקוד Bulb נוירונים חוש הריח שימוש משולב In vivo Electroporation ו Gal4 מבוסס Enhancer מלכודת קווי דג הזברה
1Department of Biology, Pace University, 2Cellular and Molecular Medicine, University of California, San Diego, 3Division of Cell Biology and Cell Physiology, Zoological Institute, Braunschweig University of Technology
ברזולוציה של זמן ומרחב של מניפולציות גנטיות קובע את הספקטרום של תופעות ביולוגיות כי הם יכולים להפריע. כאן אנו משתמשים ובזמן מרחבית בדידה
Other articles by John H. Horne on PubMed
Integration of Calcium Signals by Calmodulin in Rat Sensory Neurons
We have used the fluorescently labelled calmodulin TA-CaM to follow calmodulin activation during depolarization of adult rat sensory neurons. Calcium concentration was measured simultaneously using the low affinity indicator Oregon Green BAPTA 5N. TA-CaM fluorescence increased during a 200-ms depolarization but then continued to increase during the subsequent 500 ms, even though total cell calcium was falling at this time. In the next few seconds TA-CaM fluorescence fell, but to a new elevated level that was then maintained for several tens of seconds. During a train of depolarizations that evoked a series of largely independent calcium changes TA-CaM fluorescence was in contrast raised for the duration of the train and for many tens of seconds afterwards. The presence of a peptide corresponding to the calmodulin binding domain of myosin light chain kinase significantly increased the depolarization-induced TA-CaM fluorescence increase and slowed the subsequent fall of fluorescence. We interpret the slow recovery component of the TA-CaM signal as reflecting the slow dissociation of calcium--calmodulin--calmodulin binding protein complexes. Our results show that after brief electrical activity calmodulin's interaction with calmodulin binding proteins persists for approximately one minute.
Targeting the Zebrafish Optic Tectum Using in Vivo Electroporation
INTRODUCTION: In vivo electroporation is a method for delivery of plasmids and other oligonucleotide reagents that offers precise temporal control. In zebrafish, in vivo electroporation is particularly well-suited to delivering green fluorescent protein (GFP) expression vectors to the developing central nervous system. This protocol describes a modification of in vivo electroporation that can be used to specifically target the developing optic tectum of zebrafish embryos beginning at 24 h post-fertilization (hpf). The electroporation electrodes required for this approach can be constructed easily from relatively inexpensive materials. Microinjection of plasmid DNA to the midbrain ventricle followed by precise positioning of the electroporation electrodes allows for the targeting of developing neurons in only one hemisphere of the optic tectum. Using this protocol, the optic tectum can be effectively targeted in a high percentage (79%) of expressing embryos. This method can also be used to simultaneously deliver expression vectors and loss-of-function reagents, which can provide precise temporal control of the knockdown of gene function.
The Temporal Resolution of in Vivo Electroporation in Zebrafish: a Method for Time-resolved Loss of Function
One caveat to current loss-of-function approaches in zebrafish is that they typically disrupt gene function from the beginning of development. This can be problematic when attempting to study later developmental events. In vivo electroporation is a method that has been shown to be effective at incorporating reagents into the developing nervous system at multiple later developmental stages. The temporal and spatial characteristics of in vivo electroporation that have been previously demonstrated suggest that this could be a powerful approach for time-resolved loss-of-function analysis. Here, in an attempt to demonstrate the efficacy of this approach for analysis of a specific developmental timeframe--that of initial development of the zebrafish visual system-we have done a systematic characterization of the efficiency of in vivo electroporation in zebrafish across multiple developmental stages, from 24 to 96 h postfertilization. We show that electroporation is efficient at delivering expression plasmids to large numbers of neurons at multiple developmental steps, including 24, 48, or 96 h postfertilization. Expression from electroporated plasmids is maximal within 24 h, and significant and useful expression is seen within 6 h. Electroporation can be used to deliver two separate expression plasmids (green fluorescent protein and mCherry), resulting in coexpression in 97% of cells. Most importantly, electroporation can be used to incorporate siRNA reagents, resulting in 84% knockdown of a target protein (green fluorescent protein). In conclusion, in vivo electroporation is an effective method for delivering both DNA-based expression plasmids and RNA interference-based loss-of-function reagents, and exhibits the appropriate characteristics to be useful as a time-resolved genetic approach to investigate the molecular mechanisms of visual system development.
